Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum
- Department of Physiology and Biophysics, Case Western Reserve University, United States
- Department of Biology, University of Victoria, Canada
- Department of Biomedical Sciences, Marshall University, United States
Figures
Figure 1 with 2 supplements
Hsp47 interacts with GABAA receptors.
(A) Endogenous interactions between GABAA receptor α1 subunits and Hsp47. Mouse brain homogenates from 8 to 10 weeks C57BL/6 J mice were immunoprecipitated with an anti-α1 antibody, and the …
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Figure 1—source data 1
Original files for the western blot analysis in Figure 1A and B.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig1-data1-v2.zip
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Figure 1—source data 2
PDF containing the original blots in Figure 1A and B with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig1-data2-v2.zip
Figure 1—figure supplement 1
The GABAA receptor biogenesis pathway.
Individual GABAA receptor subunits fold in the endoplasmic reticulum (ER). Properly folded subunits assemble into a heteropentamer in the ER for subsequent trafficking to the plasma membrane. …
Figure 1—figure supplement 2
In vitro interactions between Hsp47 and membrane proteins.
(A) Recombinant His-tagged Hsp47 protein was mixed with FLAG-tagged ZIP7, hERG, GST-tagged GABAA receptor α1 recombinant proteins, or buffer only in binding buffers (50 mM Tris, pH 7.5, 150 mM NaCl, …
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Figure 1—figure supplement 2—source data 1
Original files for the western blot analysis in Figure 1—figure supplement 2A.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig1-figsupp2-data1-v2.zip
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Figure 1—figure supplement 2—source data 2
PDF containing the original blots in Figure 1—figure supplement 2A with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig1-figsupp2-data2-v2.zip
Figure 2 with 1 supplement
Hsp47 positively regulates the surface expression of endogenous GABAA receptors in cultured neurons.
(A, B) Effect of knocking down Hsp47 (A) and overexpressing Hsp47 (B) on the surface expression of endogenous GABAA receptor subunits in primary rat hippocampal neurons. Cultured neurons were …
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Figure 2—source data 1
Data used for graphs presented in Figure 2A, B and C.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig2-data1-v2.zip
Figure 2—figure supplement 1
Hsp47 expression in the central nervous system.
(A) Hsp47 and GABAA receptor β2/β3 subunit protein expression in various mouse brain regions according to SDS-PAGE and Western blot analysis. Three biological replicate experiments were performed …
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Figure 2—figure supplement 1—source data 1
Original files for the western blot analysis in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
PDF containing the original blots in Figure 2—figure supplement 1A with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig2-figsupp1-data2-v2.zip
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Figure 2—figure supplement 1—source data 3
Data used for graphs presented in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig2-figsupp1-data3-v2.zip
Figure 3 with 1 supplement
Hsp47 preferentially binds the folded conformation of GABAA receptor subunits.
(A) Overexpression of Hsp47 increases the endo H-resistant post-ER glycoform of the α1 subunit in HEK293T cells stably expressing α1β2γ2 GABAA receptors. The peptide-N-glycosidase F (PNGase F) …
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Figure 3—source data 1
Original files for the western blot analysis in Figure 3A, B, C, D, E and F.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig3-data1-v2.zip
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Figure 3—source data 2
PDF containing the original blots in Figure 3 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig3-data2-v2.zip
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Figure 3—source data 3
Data used for graphs presented in Figure 3A, B, C, D, E and F.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig3-data3-v2.zip
Figure 3—figure supplement 1
Effect of Hsp47 on the degradation of wild type GABAA receptors.
(A) Whole-cell patch clamping was performed to record GABA-induced currents. HEK293T cells expressing α1β2γ2 GABAA receptors were transfected with empty vector (EV) control or SERPINH1 cDNA …
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Figure 3—figure supplement 1—source data 1
Original files for the western blot analysis in Figure 3—figure supplement 1B, C, D, and E.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
PDF containing the original blots in Figure 3—figure supplement 1 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig3-figsupp1-data2-v2.zip
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Figure 3—figure supplement 1—source data 3
Data used for graphs presented in Figure 3—figure supplement 1A, B, and E.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig3-figsupp1-data3-v2.zip
Figure 4 with 1 supplement
Hsp47 promotes the assembly of GABAA receptors.
(A) Hsp47 overexpression increases FRET efficiency between CFP-tagged α1 subunit and YFP-tagged β2 subunit of GABAA receptors. HEK293T cells were transfected with CFP-tagged α1 subunit, YFP-tagged …
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Figure 4—source data 1
Original files for the western blot analysis in Figure 4B and C.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig4-data1-v2.zip
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Figure 4—source data 2
PDF containing the original blots in Figure 4 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig4-data2-v2.zip
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Figure 4—source data 3
Data used for graphs presented in Figure 4A, B, D and E.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig4-data3-v2.zip
Figure 4—figure supplement 1
Dose-response curves of GABAA receptors.
HEK293T cells were transfected with α1, β2, and γ2 subunits of GABAA receptors, or α1-CFP, β2-YFP, and γ2 subunits. Forty-eight hours post-transfection, whole-cell patch-clamping …
Figure 5 with 2 supplements
Hsp47 positively regulates the functional surface expression of epilepsy-associated GABAA receptors.
(A) Overexpression of Hsp47 increases the endo H-resistant post-ER glycoform of the α1 subunit in HEK293T cells expressing α1(A322D)β2γ2 GABAA receptors. PNGase F treatment serves as a control for …
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Figure 5—source data 1
Original files for the western blot analysis in Figure 5A, B, C and F.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-data1-v2.zip
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Figure 5—source data 2
PDF containing the original blots in Figure 5 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-data2-v2.zip
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Figure 5—source data 3
Data used for graphs presented in Figure 5A, B, C, D, F and G.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-data3-v2.zip
Figure 5—figure supplement 1
Effect of Hsp47 on the degradation of a GABAA receptor variant.
(A, B) HEK293T cells expressing α1(A322D)β2γ2 GABAA receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids (A), or transfected with non-targeting (NT) control siRNA or …
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Figure 5—figure supplement 1—source data 1
Original files for the western blot analysis in Figure 5—figure supplement 1A,B,C.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
PDF containing the original blots in Figure 5—figure supplement 1 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-figsupp1-data2-v2.zip
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Figure 5—figure supplement 1—source data 3
Data used for graphs presented in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-figsupp1-data3-v2.zip
Figure 5—figure supplement 2
Overexpression of Hsp47 in HEK293T cells expressing a variety of pathogenic GABAA receptor variants.
HEK293T cells expressing α1(S76R)β2γ2, α1(D219N)β2γ2, or α1(G251D)β2γ2 GABAA receptors were transfected with empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, …
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Figure 5—figure supplement 2—source data 1
Original files for the western blot analysis in Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-figsupp2-data1-v2.zip
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Figure 5—figure supplement 2—source data 2
PDF containing the original blots in Figure 5—figure supplement 2 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-figsupp2-data2-v2.zip
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Figure 5—figure supplement 2—source data 3
Data used for graphs presented in Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig5-figsupp2-data3-v2.zip
Figure 6
Genetic manipulations of Hsp47 do not activate the UPR.
(A) HEK293T cells expressing WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors were transiently transfected with non-targeting (NT) control siRNAs or siRNAs against SERPINH1 (#1 or #2). Forty-eight hours …
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Figure 6—source data 1
Original files for the western blot analysis in Figure 6A, B, C and D.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig6-data1-v2.zip
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Figure 6—source data 2
PDF containing the original blots in Figure 6 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig6-data2-v2.zip
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Figure 6—source data 3
Data used for graphs presented in Figure 6A, B, C and D.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig6-data3-v2.zip
Figure 7 with 2 supplements
Hsp47 has a general effect on increasing the surface expression of the Cys-loop receptors.
(A) HEK293T cells were transfected with nAChR subunits (α4 (CHRNA4) and β2 (CHRNB2)) and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, cells were lysed …
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Figure 7—source data 1
Original files for the western blot analysis in Figure 7A and D.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-data1-v2.zip
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Figure 7—source data 2
PDF containing the original blots in Figure 7 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-data2-v2.zip
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Figure 7—source data 3
Data used for graphs presented in Figure 7A, B, C and D.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-data3-v2.zip
Figure 7—figure supplement 1
Effect of Hsp47 on the biogenesis of α7 nAChRs.
(A) Hsp47 overexpression does not change the FRET efficiency between Cerulean (an improved CFP variant)-tagged α7 subunit and Venus (an improved YFP variant)-tagged α7 subunit of nAChRs. HEK293T …
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Figure 7—figure supplement 1—source data 1
Original files for the Western blot analysis in Figure 7—figure supplement 1B.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-figsupp1-data1-v2.zip
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Figure 7—figure supplement 1—source data 2
PDF containing the original blots in Figure 7—figure supplement 1 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-figsupp1-data2-v2.zip
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Figure 7—figure supplement 1—source data 3
Data used for graphs presented in Figure 7—figure supplement 1A, B.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-figsupp1-data3-v2.zip
Figure 7—figure supplement 2
Effect of Hsp47 on the biogenesis of structurally diverse proteins.
(A) HEK293T cells were transfected with NMDA receptor subunits (GluN1 and GluN2A (WT or M705V)) and empty vector (EV) control or SERPINH1 cDNA plasmids. Forty-eight hours post transfection, the …
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Figure 7—figure supplement 2—source data 1
Original files for the western blot analysis in Figure 7—figure supplement 2A,B,C.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-figsupp2-data1-v2.zip
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Figure 7—figure supplement 2—source data 2
PDF containing the original blots in Figure 7—figure supplement 2 with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-figsupp2-data2-v2.zip
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Figure 7—figure supplement 2—source data 3
Data used for graphs presented in Figure 7—figure supplement 2A,B,C.
- https://cdn.elifesciences.org/articles/84798/elife-84798-fig7-figsupp2-data3-v2.zip
Figure 8
Proposed mechanism of Hsp47 in the assembly of GABAA receptors.
BiP and calnexin assist the subunit folding early in the ER lumen. Hsp47 operates after BiP and binds the folded states of the α1 or β subunits in the ER lumen. Hsp47 links the α1 and β subunits and …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | HEK293T | ATCC | Cat#: CRL-3216 | |
| Cell line (Homo-sapiens) | HEK293T | Abgent | Cat#: CL1032 | |
| Cell line (Homo-sapiens) | Fibroblasts harbouring L444P β-glucocerebrosidase | Coriell Institute | Cat#: GM20272 | |
| Transfected construct (Human) | siRNA to SERPINH1 | Dharmacon | Cat#: J-011230-05-0005 | |
| Transfected construct (Human) | siRNA to SERPINH1 | Dharmacon | Cat#: J-011230-06-0005 | |
| Transfected construct (Human) | siRNA, non-targeting control | Dharmacon | Cat#: D-001810-01-20 | |
| Biological sample (rat) | E18 hippocampus | BrainBits | Cat#: SDEHP | |
| Biological sample (rat) | E18 cortex | BrainBits | Cat#: SDECX | |
| Antibody | anti-GABAAα1 (mouse monoclonal) | Millipore | Cat#: MAB339 | WB (1:2000) |
| Antibody | anti-GABAAβ2/3 (mouse monoclonal) | Millipore | Cat#: 05–474 | WB (1:1000) IF (1:250) |
| Antibody | anti-GABAAβ2 (rabbit polyclonal) | Millipore | Cat#: AB5561 | WB (1:1000) |
| Antibody | anti-GABAAγ2 (rabbit polyclonal) | Millipore | Cat#: AB5559 | WB (1:1000) |
| Antibody | anti-NeuN (rabbit polyclonal) | Millipore | Cat#: ABN78 | IF (1:500) |
| Antibody | anti-GABAAα1 (rabbit polyclonal) | R&D systems | Cat#: PPS022 | WB (1:1000) |
| Antibody | anti-GABAAα1 (goat polyclonal) | Santa Cruz Biotechnology | Cat#: SC-31405 | WB (1:1000) |
| Antibody | anti-nAChR α4 (mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-74519 | WB (1:1000) |
| Antibody | anti-GABAAα1 (rabbit polyclonal) | Synaptic Systems | Cat#: 224203 | IF (1:250) |
| Antibody | anti-GABAAγ2 (rabbit polyclonal) | Synaptic Systems | Cat#: 224003 | IF (1:250) |
| Antibody | anti-nAChR β2 (rabbit polyclonal) | Proteintech | Cat#: 17844–1-AP | WB (1:1000) |
| Antibody | anti-nAChR α7 (rabbit polyclonal) | Proteintech | Cat#: 21379–1-AP | WB (1:1000) |
| Antibody | anti-ATF6 (rabbit polyclonal) | Proteintech | Cat#: 24169–1-AP | WB (1:2000) |
| Antibody | anti-FLAG (rabbit polyclonal) | Proteintech | Cat#: 20543–1-AP | WB (1:2000) |
| Antibody | anti-Hsp47 (mouse monoclonal) | Proteintech | Cat#: 67863–1-lg | WB (1:1000) |
| Antibody | anti-5-HT3A (goat polyclonal) | Origene | Cat#: TA302602 | WB (1:1000) |
| Antibody | anti-GluN2A (rabbit monoclonal) | Abcam | Cat#: ab124913 | WB (1:3000) |
| Antibody | anti-Hsp47 (rabbit monoclonal) | Abcam | Cat#: ab109117 | WB (1:1000) IF: (1:250) |
| Antibody | anti-Grp78 (rabbit monoclonal) | Abcam | Cat#: ab108613 | WB (1:2000) |
| Antibody | anti-Na+/K+ATPase (rabbit monoclonal) | Abcam | Cat#: ab76020 | WB (1:10,000) |
| Antibody | anti-hERG (rabbit polyclonal) | ThermoFisher | Cat#: PA3-860 | WB (1:10,000) |
| Antibody | anti-XBP1s (rabbit monoclonal) | Cell Signaling | Cat#: 12782 S | WB (1:1000) |
| Antibody | anti-CHOP (mouse monoclonal) | Cell Signaling | Cat#: 2895 S | WB (1:1000) |
| Antibody | anti-His (mouse monoclonal) | Cell Signaling | Cat#: 2366 S | WB (1:1000) |
| Antibody | anti- glucocerebrosidase (rabbit polyclonal) | Sigma | Cat#: G4046 | WB (1:1000) |
| Antibody | anti-FLAG (mouse monoclonal) | Sigma | Cat#: F1804 | WB (1:2000) |
| Antibody | anti-β-actin (mouse monoclonal) | Sigma | Cat#: A1978 | WB (1:20,000) |
| Antibody | Fluorescent anti-β-actin Rhodamine | Biorad | Cat#: 12004163 | WB (1:10,000) |
| Antibody | anti- Grp78 (rabbit polyclonal) | Abgent | Cat#: AP5041c | WB (1:5000) |
| Antibody | anti-Hsp47 (mouse monoclonal) | Enzo Life Sciences | Cat#: ADI-SPA-470-F | WB (1:2000) IF (1:250) |
| Antibody | Alexa 594-conjugated goat anti-rabbit | ThermoFisher | Cat#: A11037 | IF (1:500) |
| Antibody | Alexa 594-conjugated goat anti-mouse | ThermoFisher | Cat#: A11032 | IF (1:500) |
| Antibody | Alexa 405-conjugated goat anti-rabbit | ThermoFisher | Cat#: A31556 | IF (1:500) |
| Recombinant DNA reagent | pCMV6-GABRA1-CFP (plasmid) | This paper | See Materials and Methods, Section Plasmids and siRNAs | |
| Recombinant DNA reagent | pCMV6-GABRB2 (plasmid) | Origene | Cat#: RC216424 | |
| Recombinant DNA reagent | pCMV6-GABRB2-YFP (plasmid) | This paper | See Materials and Methods, Section Plasmids and siRNAs | |
| Recombinant DNA reagent | pCMV6-GABRG2 (plasmid) | Origene | Cat#: RC209260 | |
| Recombinant DNA reagent | pCMV6-HTR3A (plasmid) | Origene | Cat#: SC122578 | |
| Recombinant DNA reagent | pCMV6-HTR3B (plasmid) | Origene | Cat#: MR206966 | |
| Recombinant DNA reagent | pCMV6-KCNH2 (plasmid) | Origene | Cat#: RC215928 | |
| Recombinant DNA reagent | pCMV6-Entry Vector (plasmid) | Origene | Cat#: PS100001 | |
| Recombinant DNA reagent | pcDNA3.1-GRIN2A (plasmid) | GenScript | Cat#: OHu24642D | |
| Recombinant DNA reagent | pcDNA3.1-GRIN1 (plasmid) | GenScript | Cat#: OHu22255D | |
| Recombinant DNA reagent | CHRNA4 (plasmid) | Addgene | RRID: Addgene_24271 | |
| Recombinant DNA reagent | CHRNA4-YFP (plasmid) | Addgene | RRID: Addgene_15245 | |
| Recombinant DNA reagent | CHRNB2 (plasmid) | Addgene | RRID: Addgene_24272 | |
| Recombinant DNA reagent | CHRNB2-CFP (plasmid) | Addgene | RRID: Addgene_15106 | |
| Recombinant DNA reagent | pcDNA3.1-CHRNA7 (plasmid) | Addgene | RRID: Addgene_62276 | |
| Recombinant DNA reagent | pcDNA3.1-CHRNA7-cerulean (plasmid) | https://pubmed.ncbi.nlm.nih.gov/23586521/ | ||
| Recombinant DNA reagent | pcDNA3.1-CHRNA7-venus (plasmid) | https://pubmed.ncbi.nlm.nih.gov/23586521/ | ||
| Recombinant DNA reagent | pCMV6-XL5-SERPINH1 (plasmid) | Origene | Cat#: SC119367 | |
| Recombinant DNA reagent | pRK5-HA-Ubiquitin (plasmid) | Addgene | RRID: Addgene_17608 | |
| Recombinant DNA reagent | psPAX2 (plasmid) | Addgene | RRID: Addgene_12260 | |
| Recombinant DNA reagent | pMD2.G (plasmid) | Addgene | RRID: Addgene_12259 | |
| Recombinant DNA reagent | pCIG3 (plasmid) | Addgene | RRID: Addgene_78264 | |
| Recombinant DNA reagent | pHRIG-AktDN (plasmid) | Addgene | RRID: Addgene_53597 | |
| Recombinant DNA reagent | pHRIG-SERPINH1 (plasmid) | This paper | See Materials and Methods, Section Plasmids and siRNAs | |
| Recombinant DNA reagent | Scrambled siRNA GFP lentivector (plasmid) | Applied Biological Materials | Cat#: LV015-G | |
| Recombinant DNA reagent | SERPINH1 siRNA lentivector (smartpool) (plasmid) | Applied Biological Materials | Cat#: 435050960395 | |
| Peptide, recombinant protein | GST | Novus | Cat#: NBC1-18537 | |
| Peptide, recombinant protein | GST-GABRA1 | Abnova | Cat#: H00002554-P01 | |
| Peptide, recombinant protein | GST-GABRB2 | Abnova | Cat#: H00002561-P01 | |
| Peptide, recombinant protein | His-Hsp47 | Novus | Cat#: NBC1-22576 | |
| Peptide, recombinant protein | FLAG-ZIP7 | Origene | Cat#: TP313722 | |
| Peptide, recombinant protein | hERG | Abnova | Cat#: H00003757-G01 | |
| Peptide, recombinant protein | His-GABRA1-ERD | MyBioSource | Cat#: MBS948971 | |
| Peptide, recombinant protein | His-GABRB2-ERD | MyBioSource | Cat#: MBS953526 | |
| Peptide, recombinant protein | Hsp47 | Abcam | Cat#: ab86918 | |
| Commercial assay or kit | GenBuilder cloning kit | GenScript | Cat#: L00701 | |
| Chemical compound, drug | AA147 | Tocris Bioscience | Cat#: 6759 | |
| Chemical compound, drug | AA263 | Sigma | Cat#: R699470 | |
| Chemical compound, drug | DAPI | ThermoFisher | Cat#: D1306 | |
| Software, algorithm | ImageJ | National Institutes of Health | https://imagej.nih.gov/ij/ | |
| Software, algorithm | pClamp10 | Molecular Devices | https://www.moleculardevices.com/products/axon-patch-clamp-system | |
| Software, algorithm | Automatic patch clamping | Ionflux Mercury16 | https://www.fluxionbio.com/ionflux-mercury-automated-patch-clamp | |
| Software, algorithm | JASCO Spectra Manager | JASCO | ||
| Software, algorithm | Monolith NT.115 | NanoTemper | ||
| Software, algorithm | Origin | Origin Lab | https://www.originlab.com/ | |
| Software, algorithm | PyMOL | Schrodinger | https://pymol.org/ |
